1. Field of the Invention
The present invention is in the field of vaccines useful for raising an immune response in an animal. In particular, the invention relates to H. influenzae polysaccharide-N. meningitidis outer membrane protein conjugates, pharmaceutical compositions and the use thereof.
2. Background Information
Haemophilus influenzae are small, pleomorphic, Gram-negative coccobacilli. Isolates are classified into six antigenically distinct capsular types (a-f) and nonencapsulated, nontypable strains. Haemophilus influenzae can cause meningitis, otitis media, sinusitis, epiglottitis, septic arthritis, occult febrile bacteremia, cellulitis, pneumonia, and empyema; occasionally this organism causes neonatal meningitis and septicemia. Other H. influenzae infections include purulent pericarditis, endocarditis, conjunctivitis, osteomyelitis, peritonitis, epididymo-orchitis, glossitis, uvulitis, and septic thrombophlebitis. Most cases of invasive diseases in children before the introduction of H. influenzae type b (Hib) conjugate vaccination were caused by type b. Nonencapsulated organisms can cause invasive disease in newborns. Nonencapsulated strains cause upper respiratory tract infection, including otitis media, sinusitis, and bronchitis, and may cause pneumonia.
The source of the organism is the upper respiratory tract of humans. The mode of transmission is presumably person to person, by direct contact, or through inhalation of droplets of respiratory tract secretions containing the organism. Asymptomatic colonization by nonencapsulated strains is frequent; organisms are recovered from the throat of 60% to 90% of children. Colonization by type b organisms, however, is infrequent, ranging from 2% to 5% of children in the pre-vaccine era, and appears to be even less frequent with widespread Hib conjugate vaccination. The exact period of communicability is unknown but may be for as long as the organism is present in the upper respiratory tract.
Before the introduction of effective vaccines, Hib was the most common cause of bacterial meningitis in children in the United States and in many other countries. Meningitis and other invasive infections were most common in children 3 months to 3 years of age and approximately half of the cases occurred in infants younger than 12 months. The age-specific incidence of invasive type b disease in different populations in countries has varied; the proportion of disease in infants younger than 12 months tends to be greatest in populations with the highest total incidence, resulting in a lower median age of cases. In contrast to meningitis and most other invasive Hib diseases, epiglottitis is rare in infants younger than 12 months; its peak occurrence in the pre-vaccine era was 2 to 4 years of age. Epiglottitis also can occur in older, unvaccinated children and adults.
Invasive disease has been more frequent in boys, African-Americans, Alaskan Eskimos, Apache and Navajo Indians, child care center attendees, children living in overcrowded conditions and children who were not breast-fed. Unimmunized children, particularly those younger than 4 years who are in prolonged, close contact (such as in a household) with a child with invasive Hib disease, are at an increased risk for serious infection from this organism. Other factors predisposing to invasive disease include sickle cell disease, asplenia, HIV infection, certain immunodeficiency syndromes, and malignancies. Infants younger than 1 year with documented invasive infection are at an approximate 1% risk of recurrence, if not subsequently vaccinated.
Since 1988 when Hib conjugate vaccines were introduced, the incidence of invasive Hib disease has declined by 95% in infants and young children and the incidence of invasive infections caused by other encapsulated types is now similar to that caused by type b. As a result of this success, the U.S. Public Health Service has targeted Hib disease in children younger than 5 years for elimination in this country. Invasive Hib disease occurs now in this country primarily in under vaccinated children and among infants too young to have completed the primary series of vaccinations.
Four Hib conjugate vaccines have been licensed in the United States. These vaccines consist of the Hib capsular polysaccharide (ie, polyribosylribotol phosphate (PRP) or PRP oligomers) covalently linked to a carrier protein directly or via an intervening spacer molecule. Protective antibodies are directed against PRP. Conjugate vaccines differ in composition and immunogenicity and, as a result, recommendations for their use differ. For example, PRP-D is recommended only for children 12 months of age and older, whereas the other three vaccines, HbOC, PRP-T, and PRP-OMP, are recommended for infants beginning at 2 months of age.
Adjuvants are substances that augment the immune response to antigens and, therefore, have been used in many vaccines and vaccine candidates. The immune stimulatory effect of adjuvants is not antigen specific, as they boost immune responses towards many different types of antigens. The only adjuvants currently approved for human use by the FDA are aluminum salts, but many adjuvants used in animal vaccinations and in newer vaccine candidates are microbial in origin (61) e.g. Freund""s adjuvant, Corynebacterium parvum, muramyl dipetide, tetanus toxoid, etc. The mechanisms for the immunopotentiating ability of microbial substances are unknown.
The major outer membrane proteins of the pathogenic Neisseria (Neisseria gonorrhoeae and Neisseria meningitidis) have been investigated for adjuvant potential (36,37,39,40,60) and for the mechanism behind their immunopotentiating ability. The proteins of interest are protein IA (PIA) and protein IB (PIB) from the gonococcus and class 1, 2 or 3 proteins from the meningococcus (C1, C2 and C3 respectively) (4). They all function as porins (41,43,62), have significant amino acid sequence homology amongst each other (6,7,21,59) and are considered to be part of the gram negative porin superfamily (26).
Neisserial porins, when complexed non-covalently with malarial peptides, were shown to enhance the antibody response to these peptides as compared to when the peptides were used as an immunogen alone or covalently linked to other proteins (39,40). In addition, peptides derived from Group A streptococcus (38), influenza virus hemagglutinin (38), or Trypanosome bruceii (40) were shown to be more immunogenic in mice when incorporated into complexes containing Neisserial porins as compared to when the mice were immunized with peptides alone. Meningococcal outer membrane vesicles (OMV), mainly consisting of the class 2 protein, were used as a carrier to boost the immune response towards the H. influenzae polysaccharide capsule in the recently licensed H. influenzae type b vaccine developed by Merck (10). Furthermore, Livingston has explored the use of purified Neisserial porins as adjuvants in anti-melanoma vaccines. Melanoma cells express much higher levels of the human gangliosides GM2 or GD3 on their surface as compared to normal melanocytes. To augment the immune response to GM2 and GD3, and possibly induce tumor immunity in melanoma patients, GM2 and GD3 were noncovalently associated with purified Neisserial porins and volunteers with malignant melanoma were immunized with these vaccine constructs. Anti-GM2 or anti-GD3 antibody responses were greatly enhanced in patients immunized with porin/GM2 or porin/GD3 complexes as compared to patients immunized with these gangliosides alone or complexed with BCG (36,37). In addition, the tumor burden in patients immunized with porin/GM2 decreased significantly (personal communication, P. Livingston).
The mechanisms by which the Neisserial porins act as adjuvants are unknown. The group from Merck (10,35,56), who developed the Haemophilus polysaccharide capsulexe2x80x94meningococcal OMV conjugate vaccine, thought that it might be due to direct T cell stimulation by the class 2 protein. They initially demonstrated that the class 2 protein could directly stimulate T lymphocytes and, therefore, they renamed the class 2 protein as the Meningococcal Immune Enhancing Protein (MIEP) (35). However, it was later shown that only denatured class 2 protein at high concentrations ( greater than 50 xcexcg) could stimulate T cells, whereas the native protein had no such effect (56). Furthermore, since the majority of the Neisserial porins are in their native configuration when used as a vaccine candidate or adjuvant, the likelihood that non-specific T cell stimulation by denatured porins accounts for their immunopotentiating ability is low.
Over the last few years, details regarding the interaction between T and B lymphocytes required for antigen recognition, lymphocyte stimulation and antibody production have been elucidated. In the current model of T lymphocyte stimulation, two sets of signals between the antigen presenting cell (APC) and the T lymphocyte have been shown to be required (24,25,51). The first signal (signal 1) is delivered via the interaction of the major histocompatibility (MHC) complex on antigenpresenting cells (e.g. B lymphocytes, dendritic cells, macrophages, etc.) and the T cell receptor on T lymphocytes. The groove on the MHC complex is usually occupied by an oligopeptide derived from processed antigens (T cell epitope). The specificity of the reaction is conferred by signal 1. The second or costimulatory signal (signal 2) is delivered by the binding of two sets of counter-receptors during the interaction between the B and T lymphocytes (FIG. 1). The activated T lymphocytes then release cytokines which in turn stimulate the effector cells, for example, causing B lymphocytes to become antibody producing cells. The induction of costimulation by the interaction of these counter-receptors has been shown to be important in tumor immunity. (1,3,8,11,51,55), the prevention of tolerance (19,45,54), and for cytotoxic lymphocyte activity (1).
The T lymphocyte counter-receptors are CD28 and CTLA-4. They are both members of the immunoglobulin superfamily (9). CD28 is present on resting and activated T cells (1,8,27,30,32,34,46), while CTLA-4 is only expressed on activated T cells (17,23,31,33,51). The level of CD28 on activated T cells is 20xc3x97higher than CTLA-4 but the affinity of CD28 for its B cell counter-receptor is much lower (31,33). The B lymphocyte counter-receptors are B7 (14,20,32,48,49) and the more recently discovered B7-2(2,12,13,16,24). B7 and B7-2 are members of the immunoglobulin superfamily (13-15) and are only present on activated B lymphocytes (14). Several lines of evidence demonstrate the relationship of the newer ligand, B7-2, to T lymphocyte costimulation; 1) CTLA-4 binding to activated B cells is only partially inhibited by an anti-B7 monoclonal antibody (mAb) (24), 2) lymphocytes derived from mice deficient in B7 expression can still costimulate T cells (12,13), 3) transfectants expressing B7-2 alone can costimulate T cells (13,16), and 4) a mAb specific for B7-2 can inhibit T lymphocyte costimulation by B cells (24) or B7-2 transfectants (13). The significance of the initially described B7 antigen as a costimulation counter-receptor is controversial because the expression of B7-2 occurs earlier than the expression of B7 and there is more B7-2 present on the surface of activated B lymphocytes than B7 (24). A schematic representation of T lymphocyte costimulation and the costimulatory counter-receptors is illustrated in FIG. 1.
There is preliminary evidence presented by various investigators that microbial products can stimulate B lymphocytes. Liu et al. have demonstrated that lipopolysaccharide (LPS), mitogenic influenza virus, and an antigen that mimics viral infection (polyinosinic-polycytidylic acid), all stimulate B lymphocytes, which in turn costimulate T lymphocytes (25). Vordermeier has demonstrated that purified Salmonella typhi porins (free of LPS) are potent B cell stimulators, but have minimal effect on T lymphocytes (57,58). In addition, meningococcal outer membrane preparations, mainly consisting of the meningococcal porins act as B cell mitogens and do not stimulate T lymphocytes (44,52,53). This evidence suggests that Neisserial porins, and possibly other gram-negative porins, might be able to stimulate B lymphocytes and increase B7-2 expression. The increased expression of B7-2 can mediate T lymphocyte costimulation and this could be a mechanism by which porins enhance the immune response to other antigens, such as the PRP polysaccharide presented here.
The present invention relates to an H. influenzae type b (Hib) polysaccharide-substantially pure, refolded meningococcal outer membrane protein (rPorB) conjugate.
The present invention also relates to a method of preparing an Hib polysaccharide-rPorB conjugate, comprising
(a) obtaining an Hib polysaccharide;
(b) oxidizing or selectively hydrolyzing said polysaccharide to generate aldehyde groups;
(c) obtaining a rPorB; and
(d) conjugating the polysaccharide containing aldehyde groups to the rPorB by reductive amination.
The present invention also relate to the conjugates obtained according to the methods of the invention. Optionally, the conjugates of the present invention may be combined with DTaP (diphtheria, tetanus, acellular pertussis vaccine).
The present invention also relates to pharmaceutical compositions comprising the conjugates of the invention, optionally comprising DTaP, and a pharmaceutically acceptable carrier.
The present invention also relates to a method of inducing an immune response in an animal to H. influenzae, comprising administering the conjugates of the invention to the animal in an amount effective to induce said immune response.
The invention relates in part to the surprising discovery that the Hib-rPorB conjugates of the invention induce substantially greater immune responses in animals compared to when tetanus toxoid and the recombinantly produced outer membrane P2 protein from H. influenzae is used as the antigenic protein. Substantially greater immunogenic responses were also obtained compared to the Hib-CRM conjugate which is commercially available from Lederle Laboratories, Division of American Cyanamide Company, Pearl River, N.Y. CRM197 is a site mutant, non-toxic variant of diphtheria toxin isolated from cultures of Cornebacterium diphtheriae C7(xcex20197). Seid, R. C. Jr. et al., Glycoconj. J. 6: 489-498(1989).
Furthermore, the conjugate of the present invention is especially useful in compositions also comprising DTAP, as immunologic interactions between the components, as well as epitopic suppression, is observed with conventional carrier proteins such as tetanus toxoid. The conjugates of the present invention overcome this serious limitation in combination vaccine compositions.